Project summary Protein phosphorylation is one of the most common and critical post-translational modifications governing signaling cascades in humans. Phosphorylation of protein kinases governs their activity and regulation. The importance of regulation by phosphorylation is further emphasized by the fact that protein kinases comprise nearly 2% of the human proteome and numerous kinases have been implicated in processes that control cell proliferation, motility, and apoptosis in healthy and diseased human cells. While identification of phosphorylation sites within the human proteome has dramatically progressed in recent years, our understanding of phosphorylation cascades is limited due to a distinct lack of knowledge of which kinases are responsible for each phosphorylation event and the specific arrangement of phosphorylation sites leading to an active kinase that phosphorylates its target substrate. Establishing direct connections of all human kinases to the phosphoproteome and revealing a systems-level diagram of human signaling networks also remain defining challenges. Since phosphorylation plays a central role in protein-protein interactions through phospho- binding domains, new approaches that can address these questions in a comprehensive and unbiased fashion are needed. Studying protein phosphorylation has been limited by the inability to generate phosphoproteins with the specificity of natural systems. Genetically encoded non-standard amino acids (NSAAs) have recently enabled site-specific incorporation of phosphoserine into proteins. We showed that a genomically recoded organism (GRO), in which all TAG stop codons were converted to TAA and the deletion of RF-1, converted TAG to an open sense codon dedicated for incorporating phosphoamino acids. Importantly, this technological breakthrough enables site-specific expression of human phosphoproteins in an engineered bacterial system (i.e., GRO containing phosphoserine orthogonal translation system, OTS). Furthermore, it provides a platform technology to address questions probing the connectivity of the human kinome and the functional landscape of phospho-binding domains. Here, we aim to further develop and apply this technology to generate optimized platforms to address functional questions surrounding the phosphoserine component of the human phosphoproteome (Aim 1). These new, enhanced platforms will enable studies to identify STE20 kinase substrates that will directly inform future research into multiple human disease pathways as well as define a general strategy to elucidate human kinase substrates (Aim 2). Finally, we aim to identify phosphorylation sites that are drivers of protein-protein interactions in general, followed by, a systematic screen of the STE20 substrates in a coordinated effort to assign biological function to a portion of the human phosphoproteome (Aim 3).